WO2011106904A1

WO2011106904A1 - Method for the assisted identification of inbred line of wuzhishan micropig and the specific primers thereof

Info

Publication number: WO2011106904A1
Application number: PCT/CN2010/000252
Authority: WO
Inventors: 冯书堂; 李奎; 牟玉莲; 杨述林
Original assignee: 中国农业科学院北京畜牧兽医研究所
Priority date: 2010-03-01
Filing date: 2010-03-01
Publication date: 2011-09-09
Also published as: US20120315636A1; US9029090B2

Abstract

The present invention provides a method for the assisted identification of inbred line of Wuzhishan micropig and the specific primers thereof. The method comprises detecting deoxyribonucleotide of the 1273 site of GenBank Accession Number DQ406743 of the pig to be detected that is G or A, and determining gene type of the pig to be detected that is GG, GA or AA; the GG gene type is a homozygote in which deoxyribonucleotide of the 1273 site of GenBank Accession Number DQ406743 is G; the AA gene type is a homozygote in which deoxyribonucleotide of the 1273 site of GenBank Accession Number DQ406743 is A; the GA gene type is a heterozygote of G and A; the pig to be detected of GG gene type is potential inbred line of Wuzhishan micropig; and the pig to be detected of GA gene type and AA gene type is the inbred line of non-Wuzhishan micropig. The method is useful for the breeding of inbred line of Wuzhishan micropig.

Description

Method for assisting identification of Wuzhishan miniature pig inbred line and its special primers

The invention relates to a method for assisting identification of Wuzhishan miniature pig inbred line and a special primer thereof. Background technique

In the early days of China's reform and opening up, when studying in West Germany from 1983 to 1984, researchers were fortunate enough to visit the Gottengen Minieture Pig Breeding Center in West Germany. It was learned that Western countries have applied large numbers of miniature pigs to animal models of human diseases. At the same time, it is an excellent research object for pig embryo engineering. Göttingen mini-pigs have been sold to many countries and regions, which has led to the idea of introducing China into development and application. Professor Schmidt, one of the small pig breeding experts in West Göttingen, is interested in China Meishan pigs. It is recommended that the Ministry of Agriculture of West Germany be listed as a special support for bilateral cooperation research. The researchers also received funding from the Ministry of Agriculture after returning home. However, for various reasons, the key is that West Germany does not easily send resources to China through cooperative research. During this period, the country experienced a large-scale investigation of livestock breed resources and found many specific breeds of livestock and poultry. WZSP is one of them, but it is on the verge of extinction (see July 1987, former researcher of the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Feng Weiqi, The investigation report of 17 people including the former researcher of Wang Zheng, Guangdong Provincial Animal Husbandry Research Institute). From the data analysis, WZSP's miniaturization characteristics and genetic stability are far better than the West German Göttingen mini-pigs, which will have a good application prospect.

Since 1987, the Institute of Animal Science of the Chinese Academy of Agricultural Sciences has worked for 20 years. Under the assistance of the Ministry of Agriculture, the Ministry of Science and Technology, and the National Natural Science Foundation of China, it has invested a total of 15 million yuan to make use of rare and endangered species in China. Wuzhishan Mini-Pig (WZSP), isolated and cultivated the world's first inbred pigs, has now obtained the 20th generation group (F ₂ .). The Wuzhishan miniature pigs were inbred by the siblings, their relatives, or their grandparents and their grandparents for more than 10 generations. The test proved that the genes were highly homozygous and the inheritance was very stable. It was named Wuzhishan miniature pig inbreds. (ZL02149030. 9). Compared with the original breeding herd, the inbred line has better temperament, clear genetic background, high homogeneity of gene, black-and-white color, good consistency of body type and coat color, and smaller body size and genetic stability than domestic and foreign varieties. Advantages, belonging to the new animal genetic resources, has been listed as the National Livestock and Poultry Genetic Resources Conservation Plant (C1101001). In human comparative medicine, such as burn skin grafting, cardiovascular model, oral dental disease, xenotransplantation and other deep-level, multi-faceted research and development and utilization results also show that it is not only excellent for pig breeding, high-end meat processing. Materials are the best choice for experimental animal model research. The WZSP inbred line will play a special role and immeasurable contribution in the study of human comparative medical research and xenotransplantation.

Invention disclosure

It is an object of the present invention to provide a method for assisting in the identification of Wuzhishan miniature pig inbreds and its specific primers.

The invention provides a method for assisting identification of Wuzhishan miniature pig inbred line and/or non-Wuzhishan mini-pig inbred line, and detecting the sequence of the genomic DNA of the test pig from the 5' end of the genus K GenBank Access i on Number DQ406743 Is the 1273 deoxyribonucleotide G or A, and the genotype of the test pig is GG, GA or AA; the GG genotype is sequence 1 from the 5' end of the 1273 deoxyribonucleotide to G Homozygous; the AA genotype is a homozygous sequence 1 from the 5' end of the 1273 deoxyribonucleotide to A; the GA genotype is their hybrid;

The pigs to be tested of the GG genotype are the candidate Wuzhishan miniature pig inbreds;

The pigs to be tested for the GA genotype and the pigs to be tested for the AA genotype were non-Wuzhishan minipig inbred lines. Determining the sequence 1 in the genomic DNA of the pig to be tested ( GenBank Access i on Number

The method of DQ406743) from the 5' end of the 1273th deoxyribonucleotide to G or A includes the following steps: genomic DNA extraction, PCR amplification, restriction enzyme digestion and electrophoresis.

The PCR-amplified primer pair satisfies the following conditions: using the pig's genome 匪 as a template

The PCR amplified product contains the sequence 1 ( GenBank Access i on Number DQ406743 )

The 1273th deoxyribonucleotide at the 5' end.

The PCR-amplified primer pair may specifically be a primer pair consisting of the nucleotide shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotide shown in SEQ ID NO: 3 of the Sequence Listing.

The restriction endonuclease used for the digestion may specifically be 73⁄4 sl. The electrophoresis may specifically be agarose gel electrophoresis. Pigs with GG genotype were detected by PCR-RFLP (7ksl digestion) with four bands, 357 bp, 204 bp, 134 bp and 67 bp. AA genotype pigs were identified by PCR-RFLP (Tasl digestion). Electrophoresis bands with sequence lengths of 82 bp, 275 bp, and 204 bp, respectively.

134bp and 67bp; GA genotype pigs, PCR-RFLP (Tasl digestion) detected six bands, the sequence length is 357bp, 82bp, 275bp, 204bp, 134bp and 67bp). The present invention also protects a primer pair for assisting in the identification of a Wuzhishan minipig inbred line and/or a non-Wuzhishan minipig inbred line, the primer pair satisfying the following conditions: The PCR amplification product containing the genomic DNA of the pig as a template contains Deoxyribonucleotide at position 1273 from the 5' end of SEQ ID NO: 1.

The primer pair may specifically be a primer pair consisting of the nucleotide shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotide shown in SEQ ID NO: 3 of the Sequence Listing.

The primer pair can be applied to prepare a kit for assisting identification of Wuzhishan miniature pig inbred line and/or non-Mt. Wushan small pig inbred line.

The invention also protects a kit for the identification of a Wuzhishan minipig inbred line and/or a non-Wuzhishan minipig inbred line containing the primer pair. The kit may further include a PCR reagent and an electrophoresis reagent.

The kit or the primer pair can be applied to assist in the identification of Wuzhishan miniature pig inbred line and/or non-Wuxianshan minipig inbred line.

The kit, the primer pair and the method are applicable to the breeding of pigs.

In the kit, the primer pair, the method and the application, the Wuzhishan miniature pig inbred line may specifically be the F _13th generation to the F ₂ . generation.

DRAWINGS

Figure 1 shows the restriction enzyme digestion of three genotypes; M: DNA molecular weight standard (100-1500bp ladder).

The best way to implement the invention

The following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Wuzhishan Miniature Pig Inbred Lines (Li Kai; Yan Yulian; Han Jianlin; Yang Shulin; Liu Wei; Yuan Xinxu; Guo Yong; Feng Shutang, Study on Genetic Variation of Inbred Familys of Wuzhi shan Miniature Pig Us ing Mi crosatel li te DNA Loc i Journal of Animal Husbandry and Veterinary Medicine, Chinese Journal of Animal and Veterinary Sc ences, 2009, 03). Example 1. Discovery of Pnas-4 Gene G1273A Polymorphism

The pigs used in the trial were as follows: 42 Guizhou mini-pigs, 49 Guangxi Bama miniature pigs and 42 heads Wuzhishan miniature pig inbred line.

1, PCR amplification

A pair of primers were designed according to the second intron sequence of the Pnas-4 gene (GenBank Accession Number DQ406743) (sequence 1 of the Sequence Listing) as follows:

Upstream primer (sequence 2 of the sequence listing): 5 ' - CTAGAACCACTCAAACCAAGCAGC-3 '; downstream primer (sequence 3 of the sequence listing): 5 ' - ATCAGGCAGGTAAAAGGATAACGG-3'. Using the above primer pairs, PCR amplification was carried out using the pig's genome 匪 as a template.

PCR reaction system: 2.0 μL 10 times reaction buffer, 1.6 μ LMgCl ₂ (2.5 mmol/L), 1 μL upstream (1 μmol/L), 1 μL downstream (10 μmol/ L), 0.4 μL dNTPs (10 mmol/L), 0.2 μL Taq S|, lwL template, dd 0 to 20 μL.

PCR amplification procedure: 95. C 3 min, 30 cycles (94 C 20 s, 62.5 ° C 30 s, 72 ° C 30 s), and finally extended at 72 ° C for 3 min.

The amplified product was identified by 1.5% agarose gel electrophoresis.

2, RFLP analysis

The enzyme digestion reaction system (ΙΟμ: IX buffer 1 μL, PCR product 5wL, restriction endonuclease 7ksl 0.5μL (5U), supplemented with 0 L of 10 L.

After 8 hours of digestion, the digestion results were examined by 1.5% agarose gel electrophoresis.

The digested products showed three bands. Some samples showed four bands of electrophoresis with sequence lengths of 357 bp, 204 bp, 134 bp and 67 bp. Some samples showed five bands of electrophoresis, with sequence lengths of 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively. The remaining samples showed six The electrophoresis bands were 357 bp, 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.

3. Cloning sequencing and sequence analysis

According to the PCR-RFLP analysis results, PCR amplification products showing samples of different electrophoresis bands were recovered and purified by agarose gel recovery kit (Tiangen Biochemical Technology Co., Ltd.), and the recovered DNA fragment was ligated to pGEM-T ( After Promega, according to the method of Cohen et al. {Proc Natl Acad Sci, 69:2110), the ligated product was transformed into E. coli DH5 α (Beijing Tianshi Times) competent cells according to the carboxy-penicillin resistance marker on the carrier. Positive clones were screened to obtain a recombinant plasmid containing the recovered fragment. The nucleotide sequences of the Τ7 and SP6 promoter sequences on the recombinant plasmid were used as primers. The sequencing results showed that the amplified fragments of different samples were 762 bp in length, and there was only one difference in deoxyribonucleotides. /A) , this deoxyribonucleotide is GenBank Accession Number DQ406743 from the 5' end of the 1273 Deoxyribonucleotide, designated G1273A. Among them, the 1273 deoxyribonucleotide is

The nucleotide sequence of the amplified fragment of G is shown in SEQ ID NO:1 of the Sequence Listing.

Based on the sequencing results and PCR-RFLP results, the genotypes were defined as follows:

If the allele of this locus is G, the homozygous genotype is GG, PCR-RFLP detection (7ksl digestion) is four electrophoresis bands, and the sequence lengths are 357 bp, 204 bp, 134 bp and 67 bp, respectively; The allele is A, its homozygous genotype is AA, and PCR-RFLP ( 7ksl digestion) is detected as five electrophoresis bands with sequence lengths of 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.

The heterozygous genotype of this locus was GA, and the PCR-RFLP (Tasl digestion) detected six electrophoresis bands with sequence lengths of 357 bp, 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.

The genomic DNA of the pig to be tested is PCR-amplified using the primer pair of the present invention to obtain a 762 bp fragment. When the deoxyribonucleotide at position 1273 of the 5' end of GenBank Accession Number DQ406743 was A, the restriction fragment endonuclease Tasl was digested and the 357 bp fragment was divided into 82 bp and 275 bp fragments.

The enzyme digestion identification of the samples of the three genotypes is shown in Figure 1.

The genotype test results of different breeds of pigs are shown in Table 1.

Table 1 Pnas-4 gene 7ksl polymorphism test results of different breeds of pigs

The results showed that there were only GG genotypes and G alleles in the Wuzhishan mini-pig inbred line, while the other alleles had the A allele.

The present invention finds a polymorphic locus. The Wuzhishan minipig inbred line only has the G allele at the polymorphic locus, while in other breeds of pigs, the A allele exists, so the polymorphic locus can be used. Screening for pigs to be tested. If the pig to be tested is the GG genotype, it can be used as a candidate for the Wuzhishan minipig inbred line. If the pig to be tested is a GA genotype or an AA genotype, it is certainly not a Wuzhishan minipig inbred. The method of the invention can be applied to the breeding of Wuzhishan miniature pig inbred line. All pigs in the tested pigs were pre-screened in advance, the non-Wuzhishan mini-pig inbreds were eliminated, and the candidate Wuzhishan mini-pig inbreds were identified and further confirmed by other methods. The invention can also be applied to detecting whether a commercially available Wuzhishan miniature pig inbred line is counterfeit. Example 2, Application of Pnas-4 Gene G1273A Polymorphism

Pig ear skin tissue samples were taken from the Wuzhishan Small Pig Inbred Line of the Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, including 15 F ₁₃ generations, 13 F ₁₄ generations, 11 F ₁₅ generations, and 14 F ₁₆ generations. 14 F ₁₇ generations, 14 F ₁₈ generations, 15 F ₁₉ generations, F ₂ . All samples from the 15th generation were treated with 75% alcohol and stored in a -2CTC refrigerator to prepare DNA.

1, PCR amplification

The primers are as follows:

PCR reaction system: 2. Ομ L 10 times reaction buffer, 1.6 μ LMgCl ₂ (2.5mmol/L)

1 μL upstream I (10 μmol/L), 1 μL downstream (10 μmol/L), 0.4 μL dNTPs (10 mmol/L), 0.2 μL Taq S|, lwL template, dd 0 Capacitance to 20 μL.

PCR amplification procedure: 95. C 3min, 30 cycles (94. C 20s, 62.5°C 30s, 72°C

30 s), and finally extended at 72 ° C for 3 min.

The amplified product was identified by 1.5% agarose gel electrophoresis.

2, RFLP analysis

The enzyme digestion reaction system (lO L) is as follows:

IX buffer 1 μL, PCR product 5 L, restriction endonuclease Tasl 0.5 μL (5U), supplemented with 0w 10μL.

After 8 hours of digestion, the results of the digestion were detected by 1.5% agarose gel electrophoresis, and the best effect picture was obtained by the gel imaging system, and the genotype was recorded.

The results are shown in Table 2.

Table 2 Genotypic test results of Wuzhishan mini-pig inbred lines from different generations

F ₁₅ 11 11 0 0 1. 000 0. 000 0. 000 1. 000 0. 000

F ₁₆ 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000

F ₁₇ 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000

F ₁₈ 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000

F ₁₉ 15 15 0 0 1. 000 0. 000 0. 000 1. 000 0. 000

F ₂ o 15 15 0 0 1. 000 0. 000 0. 000 1. 000 0. 000 The result shows that F ₁₃ to F ₂ . In the generation, only the GG genotype is available.

Industrial application

The present inventors have found that the Wuzhishan minipig inbred line is homozygous at the G1273A polymorphic locus, only the GG genotype. For other breeds of minipigs, there are GA and AA genotypes, so this polymorphic locus can be used to screen pigs for testing. If the pig to be tested is the GG genotype, it can be used as a candidate for the Wuzhishan mini-pig inbred line. If the pig to be tested is a GA genotype or an AA genotype, it is certainly not a Wuzhishan minipig inbred. The method of the invention can be applied to the breeding of the Wuzhishan mini-pig inbred line, and all the pigs in the test pig group are firstly screened, the non-Wuzhishan mini-pig inbred line is eliminated, and the candidate Wuzhishan mini-pig inbred line is found, and the combination is obtained. Other methods are further confirmed. The invention can also be applied to detecting whether a commercially available Wuzhishan miniature pig inbred line is counterfeit.

Claims

Rights request

1. A method for assisting in the identification of Wuzhishan miniature pig inbreds and/or non-Wuzhishan minipig inbred lines, which is to detect the 1273 deoxyribonucleotides from the 5' end of the sequence 1 of the genome of the pig to be tested. G is A, determining whether the genotype of the test pig is GG, GA or AA; the GG genotype is a homozygosity of sequence 1 from the 5' end of the 1273 deoxyribonucleotide to G; the AA genotype The homozygote of the 1st position deoxyribonucleotide from the 5' end of the sequence 1 is A; the GA genotype is a hybrid thereof; the pig of the GG genotype is a candidate Wuzhishan minipig inbred; The pigs to be tested of the GA genotype and the pigs to be tested of the AA genotype were non-Wuzhishan minipig inbred lines.

2. The method according to claim 1, wherein: the method for detecting whether the deoxyribonucleotide at position 1273 of the sequence 1 of the genomic DNA of the test pig is G or A from the 5' end comprises the following steps: Genomic DNA extraction, PCR amplification, restriction enzyme digestion and electrophoresis.

3. The method according to claim 2, wherein: the PCR-amplified primer pair satisfies the following conditions: PCR-amplified product using porcine genomic DNA as a template contains sequence 1 of porcine genomic DNA The 1273 deoxyribonucleotide at the 5' end.

The method according to claim 3, characterized in that the PCR-amplified primer pair is a primer set consisting of the nucleotide shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotide shown in SEQ ID NO: 3 of the Sequence Listing.

The method according to any one of claims 2 to 4, wherein the restriction endonuclease used for the digestion is 73⁄4 sl; and the electrophoresis is agarose gel electrophoresis.

6. The method according to any of claims 1 to 5, characterized in that: said Wuzhishan minipig inbred line for the first F ₁₃ to F _2. generation.

7. A primer pair for assisting the identification of the Wuzhishan minipig inbred line and/or the non-Wuzhishan minipig inbred line, the primer pair satisfying the following conditions: The PCR amplification product using the genomic DNA of the pig as a template contains the sequence 1 self. The 1273 deoxyribonucleotide at the 5' end.

8, according to claim 7, wherein the primer pair which is characterized in that: said Wuzhishan minipig inbred line for the first generation of F ₁₃ to F _2. generation.

9. A primer pair consisting of the nucleotides shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotides shown in SEQ ID NO: 3 of the Sequence Listing.

10. A kit for assisting in the identification of a Wuzhishan minipig inbred line and/or a non-Wuzhishan minipig inbred line, comprising the primer pair of any one of claims 7-9.

11. The kit according to claim 10, wherein: said Wuzhishan minipig inbred line for the first generation of F ₁₃ to F _2. generation.

12. Use of a primer pair according to any one of claims 7 to 9 for the preparation of a kit for the auxiliary identification of Wuzhishan miniature pig inbreds and/or non-Wuzhishan minipig inbred lines.

13. The use as claimed in claim 12, wherein: said Wuzhishan minipig inbred line for the first F ₁₃ to F _2. generation.

The primer set according to any one of claims 9 to 10 or the kit according to claim 10 or 11 for assisting in the identification of Wuzhishan miniature pig inbred line and/or non-Wuzhishan minipig inbred line.

15. The use of claim 14, wherein: said Wuzhishan minipig inbred line for the first generation of F ₁₃ to F _2. generation.

Use of the method according to any one of claims 1 to 6, the primer set according to any one of claims 1 to 9 and the kit according to claim 10 or 1 1 in the breeding of pigs.